Self-triggered flow cytometer

ABSTRACT

An optical spectrum analyzer ( 100 ) having a an excitation light source ( 103 ) with a luminated bio-sample ( 114 ) carried by a flow path ( 102 ). A spectrum dispersive element  213  dispersants lumineses light generated by the bio-sample ( 114 ).

CROSS REFERENCE TO RELATED APPLICATION

This application is a nonprovisional of U.S. Provisional PatentApplication No. 62/089864 filed on Dec. 10, 2014. This applicationincorporates the disclosure of such application in its entirety byreference. To the extent that the present disclosure conflicts with thereferenced application, however, the present disclosure is to be givenpriority.

FIELD OF INVENTION

The present invention generally relates to cytometers; and moreparticularly, flow-cytometers and methods of making, design, use, andintegrating, these cytometers into systems.

BACKGROUND

Generally, flow-cytometry is a method for counting, examining, sorting,measurement of and characterization of various aspects of microscopicparticles, bio-particles, bio-cells, and their derivatives to determinetheir physical and/or chemical characteristic via an optical and/orelectronic detection apparatus. The early development of aflow-cytometric system was based on impedance and was developed byWallace H. Coulter in 1953. Over the years, several developments wheremade by Mack Fulwyler, Wolfgang Gohde, and others that increased thesystem's useful acceptability into the marketplace. In today'sconventional flow-cytometry, the flow-cytometery is generally based onthe use of light in the visible spectrum.

Typicality, a flow-cytometer is an analytical instrument that emits acertain frequency or frequencies of light that are directed toward asample or samples. The light emitted from the light source excites thesample to emit a certain frequency or frequencies of light from thesurface of the sample of the sample and in some cases from inside thesample. The frequencies of light that are emitted form the surface ofthe sample and in some cases emitted from the interior of the sample arecollected and analyzed.

However, certain problems such as non-integration of certain parts intounitary whole or unitary design has made the adoption of conventionalflow-cytometric machines slow to come into the marketplace. Also,because of this poor design, conventional flow-cytometers are notcapable of accurately counting and characterizing cells, bio-cellscells, and/or biologic materials, thereby limiting the use and potentialusefulness of conventional flow-cytometric instruments. Moreover, sincethis poor design causes other problems such as manufacturing,reliability, and the like which degrades the usefulness of conventionalflow-cytometers in the marketplace. Further, the poor design ofconventional flow-cytometers limits the manufacturing capability andcost parameters which drives up costs. Thus, limiting the manufacturingcapability and making conventional flow-cytometers more expensive.

It can be readily seen that conventional flow-cytometers have severalproblems and disadvantages. Despite many potential advantages offlow-cytometery, market acceptance is limited, especially in portableand non-portable applications. Further, since some of the applicationsof flow-cytometry are high volume applications, theses problems anddisadvantages do not allow conventional flow-cytometric technology to beused so as to drive the cost of flow-cytometry lower and to be moreuseful in high volume applications. Therefore, a low costflow-cytometric system or instrument capable with high volumemanufacturability and better efficiency would be highly desirable.

SUMMARY OF THE INVENTION

In various representative aspects, the present invention provides anoptical spectrum analyzer having an excitation light source thatilluminates a bio-sample. The bio-sample luminescence's with theexcitation of the bio-sample by the excitation light source. Theluminescence is directed to a dispersal element wherein the luminescenceis spread and digitized, and stored and analyzed in a digital signalprocessing unit. The digital signal process unit is directly andintimately in communication with the initial acquisition of the of theluminescence data which allows the flow-cytometer to be used as acounter.

BRIEF DESCRIPTION OF THE DRAWING

Representative elements, operational features, applications and/oradvantages of the present invention reside inter alia in the details ofconstruction and operation as more fully hereafter depicted, describedand claimed—reference being made to the accompanying drawings forming apart hereof, wherein like numerals refer to like parts throughout. Otherelements, operational features, applications and/or advantages willbecome apparent to skilled artisans in light of certain exemplaryembodiments recited in the Detailed Description, wherein:

FIG. 1 is a schematic illustration of a conventional simplifiedflow-cytometer showing an excitation light source, a flow path, aplurality of multiple photo-detectors, multiple filters, multiple gates,and a digital signal processor, and a triggering device;

FIG. 2 is a simplified schematic illustration of an embodiment of aflow-cytometer illustrating the present invention and showing anexcitation light source a flow path, a plurality of multiplephoto-detectors, and a digital signal processor, and a triggeringdevice; and

FIG. 3 is a simplified schematic illustration of another embodiment ofthe present invention illustrating a simplified flow-cytometer showingan excitation light source, a flow path, a plurality of multiplephoto-detectors, and a digital signal processor, and a triggeringdevice.

Those skilled in the art will appreciate that elements in the Figuresare illustrated for simplicity and clarity and have not necessarily beendrawn to scale. For example, the dimensions of some of the elements inthe Figures may be exaggerated relative to other elements to helpimprove understanding of various embodiments of the present invention.Furthermore, the terms ‘first’, ‘second’, and the like herein, if any,are used inter alia for distinguishing between similar elements and notnecessarily for describing a sequential or chronological order.Moreover, the terms front, back, top, bottom, over, under, and the likein the Description and/or in the claims, if any, are generally employedfor descriptive purposes and not necessarily for comprehensivelydescribing exclusive relative position. Skilled artisans will thereforeunderstand that any of the preceding terms so used may be interchangedunder appropriate circumstances such that various embodiments of theinvention described herein, for example, are capable of operation inother orientations than those explicitly illustrated or otherwisedescribed.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before addressing details of embodiments described below, some terms aredefined or clarified.

The term “filter” is intended to mean any material or device that iscapable of filtering, impeding, or preventing certain frequencies oflight to pass thought the filter.

The term “emission detector” is intended to any device or material thatis capable of detecting and/or sensing emission from the samplematerial. Typically, an emission detector can sense or detect a varietyof frequencies. However, it should be understood that the emissiondetector can made to sense or detect on a single frequency of light or asmall group of frequencies of light. Additionally, the emission detectorand be made with a variety of emission detectors.

The term “sample” is intended to mean any material or materials, anybiologic or non-biologic material that is capable of emitting light orbeing luminescent from the sample being exited from the source.

The term “bio-sample” is intended to mean any biologic material ormaterials, is capable of emitting light or luminesing from the samplebeing exited from the source.

The term “bio-cell(s)” is intended to mean a fundamental biologic unitthat can be alive and/or dead.

The term “cell(s)” is intended to mean a fundamental unit that caneither be alive of not alive. By way of example only, but not limitedto, a virus, a mycoplasem, or the like are sometimes hard to define.

The term “luminescence spectrum” is intended to mean a spectrum ofbright lines, bands, or continuous radiation characteristics of anddetermined by a specific emitting substances subjected to a specifickind of excitation.

The term “luminescence signal capturing” is intended to mean the captureand recording of output signals from the illumination of the bio-sampleby the light source which causes the bio-sample luminance at certainfrequencies that allow certain characteristics to be identified andrecorded.

The term “excitation light source” is intended to mean any suitablelight source that is capable of illuminating a sample and emittingluminescence.

The term “flow path” is intended to mean any suitable liquid that iscapable of suspending and carrying a sample to a region wherein thesample can be illuminated by the excitation light source.

The term “digital signal processor” (DSP) (also known as a Digitalsignal device) is intended to mean a specialized microprocessor (or aSIP block), with its architecture optimized for the operational needs ofdigital signal processing. Typically, the goal of DSPs is usually tomeasure, filter and/or compress continuous real-world analog signals.

As used herein, the terms “comprises”, “comprising”, or any variationthereof, are intended to reference a non-exclusive inclusion, such thata process, method, article, composition or apparatus that comprises alist of elements does not include only those elements recited, but mayalso include other elements not expressly listed or inherent to suchprocess, method, article, composition or apparatus. Other combinationsand/or modifications of the above-described structures, arrangements,applications, proportions, elements, materials or components used in thepractice of the present invention, in addition to those not specificallyrecited, may be varied or otherwise particularly adapted by thoseskilled in the art to specific environments, manufacturingspecifications, design parameters or other operating requirementswithout departing from the general principles of the same.

Further, unless expressly stated to the contrary, “or” refers to aninclusive or and not to an exclusive or. For example, a condition A or Bis satisfied by any one of the following: A is true (or present) and Bis false (or not present), A is false (or not present and B is true (orpresent, and both A and B are true (or present) Also, use of the “a” or“an” are employed to describe elements and components of the invention.This is done merely for convenience and to give a general sense of theinvention. This description should be read to include one or at leastone and the singular also includes the plural unless it is obvious thatit is meant otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

To the extent not described herein, many details regarding specificmaterials, processing acts, and circuits are conventional and may befound in textbooks and other sources within the fuel cell and chemicalarts.

Referring now to FIG. 1, FIG. 1 is conventional flow-cytometer 100incorporating an optical spectrum analyzer to analyze bio molecules thatare excited to luminescence, indicted by arrows 109 and 111. Suchluminescence's can be generated either by auto-luminescent from the cellitself or from a color label tag, or marker, attached to the bio cell orcells. Information about the bio cell or cells can be obtained thoughthe analysis of spectrum, which includes its wavelength distribution andintensity different spectrum distribution indicate the presence ofdifferent bio-cells and/or markers on the bio-cell(s). Additionally, itscorresponding intensity indicates the concentration of such distinct biocells or distinct marker on the bio-cell(s).

Typical bio-samples 114 may include varieties of bio-cell molecules,each corresponding to a unique spectral distribution through eitherauto-luminescence generation or a labeled color tag. Conventionalflow-cytometer 100 incorporates multiple photo-detectors 128, 130, 132,134, and 126, such as photomultiplier tubes (PMTS), with filters 138,140, 142, and 144 which can be customized to receive the correspondentluminescence spectrum 109 and a plurality of spectrum 111 and theirintensity and reject the rest, as shown in FIG. 1. One photo-detectorsuch as photo-detector 132 thus corresponds to one type of bio-cellmolecule or marker on the bio-cell. Conventional flow-cytometer 100includes a liquid flow path 102 containing bio-cell samples 114.Excitation light source 103, such as a semiconductor laser or LED, isused to excite bio samples 114 in region 107 on flow path 102. Thebio-cells at region 107 emit luminescence 111, which is collected bymultiple photo-detectors 131-134 and 126 with photo-detectors 128, 130,132, and 134 having correspondent filters 138, 140, 142, and 144 toallow only interested luminescence spectral contents to be detected byany individual photo-detector within 128, 130, 132, and 134. The outputanalog signals of the photo-detector 128, 130, 132, and 134 aredigitized by the digital signal processing (DSP) 141 circuitry, whichincludes multiple A/D converters. Scattering light 109 from thebio-sample region 107 is captured by a photo-detector 126, whichgenerates triggering signal 143 to trigger DSP 141 to synchronize theluminescence signal capturing. This also allows counting of the biocells or markers on the bio cells in addition to recording their emittedluminescence under light excitation. Additionally, crosstalk betweendifferent markers is compensated by complicated calibration procedures.

Different bio-cell molecules emit different characteristic luminescencedue to its own unique auto-luminescence or the attached color labels.The luminescence spectrums may overlap with each other if they are tooclose to each other in wavelengths. This crosstalk limits the detectionsensitivity and the simultaneous detection of multiple types of biocells or markers on the bio cells.

Referring now to FIG. 2, FIG. 2 is a simplified schematic illustrationof a flow-cytometer 200 that has been simplified so as to betterillustrate the present invention. FIG. 2 shows an excitation lightsource 203, a flow path 201, a plurality photo-detectors 215, withindividual photo-detectors 205, 221, 222, 223, 224, 225, 226, 227, 228,and 229 individually identified, a digital signal processor 241, and atriggering device 243. Further, as shown in FIG. 2, flow-cytometer 200includes a liquid flow path 201 having a plurality of bio-samples 214,illustrated by individual bio-samples 216, 218, and 220. The pluralityof bio-samples 214 are typically suspended in any suitable liquid,suspension, or liquid medium capable of carrying the plurality ofbio-samples 214 to region 207.

Generally, any suitable liquid suspension or liquid medium can be usedsuch as, but not limited to, saline, five percent dextrose, bufferingsolution, or the like. As illustrated in FIG. 2, flow path 201 isindicated by a plurality of flow lines 204, identified individually andin part by flow lines 106, 108, and 110.

As shown in FIG. 2, excitation light source 203 is used to illuminateregion 207 wherein individual bio samples 114 are illuminated as thebio-samples pass though region 207. Excitation light source 203 can bemade of any suitable device such as, a light emitting diode, lightemitting photo-diode, a laser, a semiconductor laser, or the like.Moreover, excitation light source 203 can be configured to produce anysuitable wavelength or wavelengths of light that is desired. By way ofexample, after the plurality of bio-samples 215 are illumined byexcitation light source 203, the plurality of bio-samples luminescence(emit light at a different wavelength, generally a lower wavelength).

Typically, the emitted light from the luminescence of the bio-sample iscollected by any suitable photo-detector or group of photo-detectors. Asshown in FIG. 2, individual photo-detectors 205, 221, 223, 225, 227,228, 230, 231 and 232 can be configured so as to capture lightindividually or can be configure so as to capture light as a pluralityof photo-detectors 215 depending upon the specific application and/orneed.

Any suitable photo-detector can be used such as, but not limited to, asemiconductor photo-detectors, Active-pixel sensors (APSs),Charged-coupled devices (CCD), or the like.

As shown in FIG. 2 an array of photo-detectors 215 can be used to recordfull bio-molecule spectrum 211 dispersed spatially by a dispersiveelement 213, such as a grating or a prism, as shown in FIG. 2.Luminescence spectrum of a color label or auto luminescence typicallyfollows its own characteristic profile that can be pre-calibrated. Whena bio-sample including multiple types of bio-cells is excited at region207 by excitation light source 203, the characteristic luminescenceprofiles will overlap, and the plurality of photo-detectors 215 orphoto-detector array 215 will record the combined luminescence spectrums211 through the individual photo-detectors 221, 222, 223, 224, 225, 226,227, 228, and 229. Additionally, it should be understood that anysuitable sized array could be used depending upon the specificapplication. Each photo-detector component receives a particularspectrum content of the spectrum distribution. As the characteristicluminescence spectrum of any particular color label andauto-luminescence has already been pre-calibrated, the distributionintensity for each bio cell can be de-convoluted from the combinedspectrums through digital data processing after the received analogsignals are converted into digital data by the DSP 241. This will allowthe use of more color labels closely spaced between each other in peakwavelength, thus increasing the types of bio-cells to be detectedsimultaneously without sacrificing the detection sensitivity. In thisdetection method, conventional hardware filters are not needed as thefull spectrum intensity will be extrapolated and thus, a software filtercan be implemented in data processing to obtain any data equivalent tothat from a hardware filter in a conventional flow-cytometer. Softwareimplementation is flexible and can be automated, thus simplifying theoverall system and reducing both the manufacturing and the service cost.

In the array detection scheme, the data converted by DSP 241 issynchronized by the conventional way, i.e. by scattered excitation lightor luminescence 209, which is received by a separate photo-detector 205to generate trigger signal 243. Bio samples 214 are usually complicatedand include varieties of bio cells and particles, which will all scatterlight. Some of those events are of interest and others are not. Whenusing scattered light as a trigger for the array signal capturesynchronization, all of those events are counted which increases thedata storage space size and slows down the data analysis. When twoevents of interest are spaced too close in time during the analysis,those events will be discarded because the system cannot distinguishthem as two separate valid events. Alternatively, luminescence signalfrom a separated photo-detector has been used as the trigger, and onlyevents of interest are captured, thus improving the data analysisthroughput. However, there are two deficiencies in this approach:

a When two events of interest are spaced too close to each other intiming, the system will miss one valid event.

b Extra photo detector for luminescence detection increases the systemmanufacturing cost.

It is the purpose of this invention to correct the deficiencies ofexisting flow cytometer by using received florescence light from thearray photo-detector to trigger the data capture and analysis. Thisself-triggering is possible because array photo-detector is designed toreceive full luminescence signals without any optical filtering. When avalid bio cell or particle is excited by a light source, it will emitauto luminescence as the background in addition to the luminescence fromthe attached color label. Conventional flow cytometer removes certainunwanted luminescence using an optical filter, and thus, makingself-triggering not practical. With the array photo-detector thatcaptures complete optical spectrums, self-triggering becomes feasible.

Referring now to FIG. 3, FIG. 3 shows a simplified schematicillustration of another embodiment of the present invention illustratinga simplified flow-cytometer 300 showing an excitation light source 303,a flow path 301, a plurality of multiple photo-detectors 315, and adigital signal processor 341, and a triggering device 343.

As shown in FIG. 3, flow-cytometer 300 shows an excitation light source303 that excites bio-samples 314 in region 307 in the flow path 301,which emit luminescence. A dispersive element 313, such as grating orprism, spreads the luminescense spectrum 311 onto array photo-detector315 with array components 321, 322, 323, 324, 325, 326, 327, 328, and329. The array photo-detector 315 can be made of avalanche photodiode(APD) or the normal PMT. An add-logic in DSP 341 after the arrayphoto-detector integrates all signals received from the array elements,and the output signal ΣDn is used as trigger 343 to synchronize the datacapturing by DSP341. This output ΣDn from the add-logic should be insynch with the excited laser source whenever it encounters a bio cell ofinterest or marker on the bio cells in the flow path as the interestedbio cell will emit either auto-luminescence or luminescence from theattached color label, thus enabling the cell counting of the events ofinterest while recording the associated luminescence spectrum for eitherin-situ or after-test data processing. More sophisticated triggering canbe adopted through logic-add of signals from selected array receivercomponents, thus allowing to target selected cells for analysis,improving system efficiency and reduce detection error when analyzingsuch cells. For example, if only certain bio cells are of interestduring analysis and the color labels for these cells are known, onlysignals from the correspondent photo receiver array components will belogic added as signal trigger during the operation.

We have thus provided a simple and effective low cost self-triggeredflow cytometer to capture complete spectrum information and improve thethroughput of cell counting while also allowing the operation inpresence of large unwanted molecules without bio-sample washing andlysing, thus simplifying the sample preparation process

The following descriptions are of exemplary embodiments of the inventionand the inventors' conceptions of the best mode and are not intended tolimit the scope, applicability or configuration of the invention in anyway. Rather, the following Description is intended to provide convenientillustrations for implementing various embodiments of the invention. Aswill become apparent, changes may be made in the function and/orarrangement of any of the elements described in the disclosed exemplaryembodiments without departing from the spirit and scope of theinvention.

In the foregoing specification, the invention has been described withreference to specific exemplary embodiments; however, it will beappreciated that various modifications and changes may be made withoutdeparting from the scope of the present invention as set forth in theclaims below. The specification and figures are to be regarded in anillustrative manner, rather than a restrictive one and all suchmodifications are intended to be included within the scope of thepresent invention. Accordingly, the scope of the invention should bedetermined by the claims appended hereto and their legal equivalentsrather than by merely the examples described above. For example, thesteps recited in any method or process claims may be executed in anyorder and are not limited to the specific order presented in the claims.Additionally, the components and/or elements recited in any apparatusclaims may be assembled or otherwise operationally configured in avariety of permutations to produce substantially the same result as thepresent invention and are accordingly not limited to the specificconfiguration recited in the claims.

Benefits, other advantages and solutions to problems have been describedabove with regard to particular embodiments; however, any benefit,advantage, solution to problems or any element that may cause anyparticular benefit, advantage or solution to occur or to become morepronounced are not to be construed as critical, required or essentialfeatures or components of any or all the claims.

We claim:
 1. An optical spectrum analyzer comprising: an excitationlight source; an illuminated bio sample carried by a flow path, thebio-sample luminescences; a spectrum dispersive element, wherein atleast a portion of the luminescence is directed to the spectrumdispersive element, wherein the luminescence is dispersed; a photodetection device having at least one photo detection input signal and atleast one photo detection output, wherein a least a portion of thedispersed luminescence is inputted into the photo detection device; adigital signal processor, the storage space having a digital storagespace input and a digital storage space output; an analysis space; and adigital trigger coupled to the photo detection output and when a signalis sensed wherein the, digital storage, and the digital processing unitstores and analyses data from the photo detection device.
 2. The opticalspectrum analyzer as claimed in claim 1 wherein the excitation lightsource is a laser diode.
 3. The optical spectrum analyzer as claimed inclaim 1 wherein the photo-detector unit (device) is an avalanche photodiode array (APD).
 4. The optical spectrum analyzer as claimed in claim3 wherein the avalanche photo diode array (ADP) includes photo receivingelements ranging from six (6) to thirty-four (34).
 5. The opticalspectrum analyzer as claimed in claim 1 wherein the digital processingunit starts to store data when triggered by received data from the arrayphoto detection unit.
 6. The optical spectrum analyzer as claimed inclaim 5 wherein all the data or a selected number of elements in thephoto detection array is a logical add.
 7. The optical spectrum analyzeras claimed in claim 1 wherein the bio sample emits auto-luminescence. 8.The optical spectrum analyzer as claimed in claim 1 wherein thebio-sample emits luminescence from marker on the bio-cell.
 9. Theoptical spectrum analyzer as claimed in claim 1 wherein the spectrumdispersive unit is a grating.
 10. The optical spectrum analyzer asclaimed in claim 1 wherein the spectrum dispersive element is a prism.11. A bio-counting device comprising: an excitation light source; anilluminated bio sample carried by a flow path; a spectrum dispersiveelement; a photo detection device having at least one photo detectioninput signal and at least one photo detection output; one photodetection output signal; a digital storage space having a digitalstorage space input and a digital storage space output; an analysisspace; and a digital trigger coupled to the photo detection output andwhen a signal is sensed, wherein the digital storage space, and thedigital processing unit stores and analyses data from the photodetection device.
 12. The bio-counting device as claimed in claim 11wherein the excitation light source is a laser diode.
 13. Thebio-counting device as claimed in claim 11 wherein the photo-detector isan avalanche photo diode array (APD).
 14. The bio-counting device asclaimed in claim 13 wherein the avalanche photo diode array (APD)includes photo diode elements ranging from six (6) photo diodes tothirty four (34) photo diodes.
 15. The bio-counting device as claimed inclaim 14 as claimed in claim 11 wherein the digital processing unitstarts to store data when triggered by received data from the arrayphoto detection unit
 16. The bio-counting device as claimed in 15wherein all the data or a selected number of elements in the photodetection array is a logical add.
 17. The bio-counting device as claimedin 15 wherein the bio-sample emits auto-luminescence.
 18. Thebio-counting device as claim 11 wherein the bio-sample emitsluminescence from markers on the bio cell.
 19. The bio-counting deviceas claim 11, wherein the spectrum dispersive element is a grating.
 20. Aselective self-triggered flow-cytometer consisting of an excitationlaser diode; an luminating bio sample carried by a flow path and excitedby the laser diode; a spectrum dispersive element dispersingluminescence from the illuminating bio sample; and an avalanchephotodiode array detecting the luminance spectrum; and analysis thebio-cell using data from the avalanche photodiode array when triggeredby the logic add of the correspondent number of elements of the photodiode array.
 21. A selective self-triggered flow-cytometer as claimed inclaim 20, wherein the avalanche photodiode array is made, in part, ofphoto receiving elements from eight (6) to elements to thirty (34).